ES2556765T3 - Nutritional support to prevent or moderate bone marrow paralysis or neutropenia during anticancer treatment - Google Patents

Abstract

Immunonutritional composition comprising: at least one immunity-enhancing agent and a pharmaceutically acceptable carrier, wherein said immunity-enhancing agent includes arginine, to prevent or temporarily reduce the toxic effect on the bone marrow caused by the anti-cancer treatment, so that said immunonutritional composition is used as part of a neoadjuvant treatment and is administered enterally.

Description

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As used herein, "prolonged administrations" are preferably continuous administrations for more than 6 weeks.

As used herein, "microorganism" includes bacteria, yeasts and / or fungi, a cell culture medium with the microorganism or a cell culture medium in which the microorganism has grown.

As used herein, a "prebiotic" is preferably a food substance that promotes the selective growth of beneficial bacteria or that inhibits the growth of pathogenic bacteria in the intestines. They are not inactivated in the stomach and / or small intestine or absorbed in the gastrointestinal tract of the person who ingests them, but are fermented by gastrointestinal microflora and / or by probiotics. Prebiotics are defined, for example, in Glenn R. Gibson and Marcel B. Roberfroid, Dietary Modulation of the Human Colonic Microbiota: Introducing the Concept of Prebiotics, introduction of the concept of prebiotics, J. Nutr. 1995 125: 1401-1412.

As used herein, probiotic microorganisms (hereinafter "probiotics") are preferably microorganisms (living, semi-viable or attenuated and / or non-replicating), metabolites, microbial cell preparations or microbial cell components that may benefit host health when administered in adequate amounts, in particular, that beneficially affect a host by improving its intestinal microbial balance and producing effects on the health or well-being of the host (Salminen S, Ouwehand A. Benno Y. and others, “Probiotics : how should they be defined ”[Probiotics: how they should be defined] Trends Food Sci. Technol. 1999: 10 107-10). It is generally believed that these microorganisms inhibit the growth and / or metabolism of pathogenic bacteria in the intestinal tract or influence it. Probiotics can also activate the host's immune function. For this reason there have been many different approaches to include probiotics in food products.

As used herein, "temporary administrations" are preferably continuous administrations of less than 6 weeks duration.

As used herein, "enteral feeding" preferably refers to complete or incomplete nutritional products that are administered to an animal's gastrointestinal system by a route other than oral administration, including without exclusion the nasogastric tube, the orogastric tube, the tube. gastric, jejunostomic probe (J probe), percutaneous endoscopic gastrostomy (PEG), access points such as a thoracic port, which provides access to the stomach, jejunum, and other appropriate access ports.

All dosage ranges mentioned in this patent application include all numbers, integers or fractions, contained in that interval.

As used herein, the term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans, nonhuman primates, rodents and the like, intended to be the recipient of a treatment. concrete. Referring to a human subject, the terms "subject" and "patient" are normally interchangeable. "Subject" may also designate a cancer patient who is experiencing apoptosis induced by anticancer therapy, either during or after such treatment.

As used herein, the terms "treatment", "treat" and "relieve" preferably refer to both prophylactic or preventive treatment (which prevents and / or slows the development of a condition or pathological disorder to which the treatment is directed) as a curative, therapeutic or disease modifier, including measures that cure, slow down, reduce symptoms and / or stop the progression of a diagnosed disease or condition, and to the treatment of patients who are at risk of contracting a disease or supposedly they have contracted, and from patients who are sick or who have been diagnosed as having a disease or medical condition. The terms "treatment", "treat" and "relieve" also refer to the maintenance and / or promotion of health in an individual who does not suffer from any disease, but may be susceptible to developing a health problem such as an imbalance of nitrogen or muscle loss The terms "treatment", "treat" and "relieve" also include potentiation or intensification of one or more primary prophylactic or therapeutic measures.

In the claims "or" is used to mean "and / or", unless explicitly stated to refer only to alternatives or mutually exclusive alternatives, although the disclosure supports a definition referring only to alternatives and "and / or" .

Throughout this patent application the term "approximately" is used to indicate that a value comprises the standard deviation of the error of the device or method used to determine said value.

It is specifically contemplated that any embodiment described in the examples is included as the embodiment of the present invention.

The terms "one" and "one", used in conjunction with the phrase "comprising" in the claims or in the description, mean one or more, unless specifically mentioned.

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Immunonutritional agents or immunonutrients are dietary components that have specific effects on the immune system or may provide additional benefits to patients suffering from the adverse consequences of starvation, disease or surgery and of apoptosis induced by anticancer therapy. It is known that these agents stimulate immune function when administered to patients enterally or parenterally and that they may be effective in improving outcomes during the preoperative period or prior to anticancer treatment and to decrease the possibility of postoperative infections and hospital stay. . As commercially available examples of immuno-nutritional regimens of enteral administration with immunity enhancing effects are: Impact® (Novartis Nutrition, Minneapolis) and Immune-Aid® (McGaw, Inc, Irvine CA), Immunex-Plex® (Victus, Inc. , Miami, FL) and AlitraQ® (Ross Laboratories, Columbus OH). These regimens contain key nutrients such as glutamine, w-3 fatty acids, arginine and / or ribonucleic acid in commercially available formulations with various quantitative compositions. The effects of these main nutrients are summarized in Table 1 of Heys, S.D. and others, Nutr. Hosp. 19: 325-332, 2004. Immunonutrients can be incorporated into current nutritional formulations for patients who have undergone cancer, eg gastrointestinal cancer surgery and pancreatic cancer surgery, or who have undergone anti-cancer therapy or are in the process of undergoing such surgery or treatment. Braga, M. et al., Nutritional Therapy & Metabolism, 24: 115-119, 2006; McCowen, K.C. and others, Am. J. Clin. Nutr. 77: 764-770, 2003; Słotwiński, R. and others, Centr. Eur. J. Immunol., 32 (3): 147-154, 2007. The formulations are administered to cancer patients preferably enterally. They can be given before, during and after the intervention or before, during and after anticancer therapeutic treatment, although studies have indicated that preoperative and perioperative immunonutrient supplementation is more effective than postoperative treatment to improve the clinical outcome of patients with gastrointestinal cancer When immunonutrition was facilitated in the postoperative period, the results were opposite, probably because the amount of substrates provided to cancer patients in the first five days after the intervention did not reach the adequate concentration in tissue and plasma quickly enough. To be active In fact, it takes about 5 days for immunonutrients to enter host tissues and therefore to modulate inflammatory mediators and fatty acid profiles. Braga, M. and others, cited above; McCowen, K.C. and others, cited above. However, to date there is still no answer to questions about the immunomodulatory effects of immunonutrition in a cancer patient, administered during the pre-, peri-or postoperative period.

The term "immunity enhancing agent" or "immunonutritional" entails the administration of specific nutritional compounds that have qualitative, enhancer or augmentative qualitative properties of the general immune system of patients undergoing anticancer or antitumor therapy or of patients whose immune function is impaired, in order to alter the cytotoxic effects induced by the tumor, improve clinical outcomes and also preserve and intensify the innate and adaptive immune processes of the host immune system to activate the removal of tumor cells in response to the induction of immunogenic determinants, as exemplified above. Examples of immunity enhancing nutritional compounds include amino acids such as L-arginine, citrulline, cysteine, glutamine, threonine, omega-3 fatty acids and nucleotides. Other examples of immunity enhancing agents include a probiotic, a probiotic biomass, a non-replicating organism, a protein source, a fatty acid, an amino acid, a nucleic acid, potassium, uric acid, a single chain oligonucleotide, a standard. molecular pathogen / microbe associated (PAMP / MAMP), a correlated compound of active hexose, carotenoids, a vitamin D receptor, branched chain amino acids, theanine, vitamin E, essential fatty acids such as EPA and DHA or EPA / DHA.

Immunity enhancing nutritional compositions can be administered by intragastric feeding.

As used herein, the term "peri-operative period" refers to the time corresponding to a patient's surgical procedure, which usually includes admission, anesthesia, surgery and recovery. Perioperative refers in general to the three phases of the intervention: preoperative, intraoperative and postoperative. The objective of peri-operative care is to provide better conditions for patients before, during and after the operation, including neoadjuvant treatment. Similarly, the pre-, peri-and post-treatment therapeutic anti-cancer refers to the period of time before, during and after chemotherapy or anti-cancer radiotherapy.

As used herein, the term "neoadjuvant" or "neoadjuvant treatment" refers to an attempt to make a tumor / tumor more sensitive to a more aggressive treatment, for example centralizing the tumor (reduction projects) and / or contracting it. , and reduce the risk of planting cancer cells during surgical removal.

As used herein, the term "aggressive treatment" refers to surgical treatments, including traditional surgery and radiosurgery, and chemotherapeutic, hormonal and radiotherapeutic treatments.

In accordance with the present invention the mechanism of cell death or apoptosis is induced by chemotherapy or radiotherapy. Cell death due to apoptosis induced by these treatments is immunogenic because all tumor cells are exposed to cell stress before death.

The transient increase in antigenicity or immunogenicity occurs in tumor cells exposed to cell death induced by anticancer therapy. The immunonutritional compositions according to the present invention

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they act more preferably on all the immune cells of the subject to preserve their immunocompetence during the stress caused by the treatment, without excluding the possibility that nutrients such as glutamine intensify the expression of HSP in stressed tumor cells, further increasing their immunogenicity . In general, apoptosis is a type of cell death that is not efficient in provoking an innate adaptive immune response. However, in some cases apoptotic cell death can be communicated with the expression of "danger signals" and therefore have a stimulating capacity of the immune system. In addition, the potentially generated immune response during this specific time can counteract the tolerogenic response that the tumor induces on its own behalf to avoid the immune response.

Therefore, a strategy used in immunotherapy is to avoid immunotolerance that can be initiated by the processing and presentation of tumor antigens by non-activated antigen presenting cells, eg dendritic cells. Some studies have shown that with agonists such as CpG oligodeoxynucleotides (ODNs) and other nucleotides, RNA, DNA and other danger signals the anti-cancer immune reaction can be stimulated better.

CpG ODNs stimulate cells that express the Toll-like receptor 9, which initiates an immunomodulation cascade that leads to the activation of B and T lymphocytes, natural killer cells, monocytes, macrophages and dendritic cells. CpG ODNs improve the host's resistance to infections and induction of innate and adaptive immune responses. Klinman, D.M. and others, Expert Opin. Biol. Ther., 4 (6): 937-946, 2004.

In addition, dendritic cells (DC) can exert a more primitive immune cellular function, that is, the ability to destroy transformed cells (cancer). This function has been ascribed to a type of dendritic cell mentioned by others as a killer dendritic cell (KDC). The KDC has the ability to kill tumor cells through various mechanisms that prevent the escape of tumor cells "resistant" to a single mechanism of death.

The dysfunction of the double function of DC during treatment, that is, the presentation of antigens and the removal of tumor cells, can be avoided by activating the population of DC through immunonutritive interventions. The combined method of an anticancer treatment such as chemotherapy and / or radiotherapy with immunonutrition can preserve an immune competence that provides a potential benefit to make the cycles more efficient, improve tolerance to immunological toxicity of treatments that can damage mucous membranes (mucositis) and produce a higher incidence of infections.

Natural killer cells and natural killer T cells are also involved in the innate cell destruction of tumor cells. Its functional capacity is greatly reduced during anticancer treatment. But to fulfill their function these cells have to maintain their ability to be activated and go through the cell cycle in order to expand their population.

Selected probiotics and other molecular patterns associated with microbes (MAMPs) have the ability to stimulate this cell population and therefore produce the removal of tumor cells.

The cytotoxic CD8 + lymphocytes (CTL) that recognize specific antigens on the cell target are depleted during the presentation of antigens to initiate the immunological reaction and in addition the treatment represses its cytotoxic acting ability. Amino acids such as glutamine, arginine and citrulline are capable of enhancing the metabolic pathways generated by the cytotoxic molecules produced by CTLs, contributing to the elimination of tumor cells when tumor antigens are more directly exposed due to induction of the cell death during treatment.

The immunonutritional compositions according to the present invention preferably comprise at least one probiotic or a combination of probiotics. Probiotics are live microorganisms that when administered in adequate amounts benefit the health of the host. Probiotics can be obtained commercially or can be produced in general by a fermentation process and optionally dried. Specific strains usually prefer particular media or substrates, which the experienced person already knows. The microorganisms may be dry or, for example, in the form of spores, in the case of spore-forming microorganisms. The drying of microorganisms after producing them by fermentation is known to the specialist. See, for example, EP 0 818 529 (Société Des Produits Nestlé), which describes a spray drying process,

or WO 0144440 (INRA). Usually bacterial microorganisms are concentrated from a medium and dried by atomization, in a fluid bed, by lyophilization (freeze drying) or other appropriate drying process. For example, microorganisms are mixed with a support material such as a carbohydrate, for example sucrose, lactose or maltodextrin, a lipid or a protein, for example milk powder, during or before drying. However, it is not necessary for the microorganisms to be in dry form. It may also be suitable to mix them directly after fermentation with a powdered nutritional composition, for example, and then optionally carry out a drying process, preferably at low temperatures (below 70 ° C). Such a method is disclosed in WO 02065840 (Société Des Produits Nestlé).

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A selected probiotic can be a strain of Bifidobacterium or Lactobacillus. Preferably it is a Bifidobacterium lactis (German crop collection: DSM20215), a Bifidobacterium longum (CNCM I-2170), a Lactobacillus paracasei (CNCM I-2116, CNCM I-1292), a Lactobacillus johnsonii (CNCM I-1225), a Lactobacillus salivarius, a Lactobacillus reuterii or mixtures thereof.

The term "probiotic" also includes non-replicated (dead) probiotic bacteria, fermentation substrate and / or probiotic derived material. The immunonutritional compositions of the present invention may contain dead or heat killed probiotics in the case of severely immunocompromised patients.

In general, it is assumed that the activation of protective immune responses by CD8 + T cells is only achieved with live vaccines. However antigens from dead bacteria were introduced into the main histocompatibility class 1 complex pathway and thus recognized by CD8 + T cells. Stimulation of protective CD8 + T lymphocytes by vaccination with non-living bacteria. Szalay, G. and others, Proc. Natl Acad. Sci. USA, 92 (26): 12389-12392, 1995.

Lactobacilli such as Lactobacillus casei have been shown to prevent intestinal infections and stimulate IgA in malnourished animals. During the different feeding periods, IgA producing cells and T lymphocytes (LT) also increased in the large intestine. The increase in IgA would indicate that the mechanisms by which probiotics inhibit tumor development may consist of a decrease in the inflammatory response. On the other hand, yogurt, in the form of a probiotic mass, not only carries the types of bacteria Streptoccus thermophilus and Lactobacillus bulgaricus, but also bifido-bacteria and sometimes Lactobacillus casei. Yogurt can inhibit the growth of intestinal carcinoma by increasing the activity of IgA, T cells and macrophages. Perdigon, G. et al., J. Dairy Sci., 78 (7): 1597-1606, 1995.

The daily dose of probiotics added to the immunonutritional compositions of the present invention may vary in the range of 107 to 1010 CFU (colony forming units).

The term "correlated active hexose compound (AHCC)" refers to a mixture of polysaccharides, amino acids, lipids and minerals derived from co-cultured mycelia of several species of basidiomycete fungi. AHCC has been linked to immunomodulation and infection protection. AHCC can increase antitumor immune surveillance by regulating both innate and adaptive immune responses (Gao, Y. and others, Cancer Immunol. Immunother., 55 (10): 1258-1266, 2006; Ritz, BW et al., J. Nutr 136: 2868-2873, 2006). The AHCC is commercially supplied by Amino Up Chemical Co. Ltd, Japan. AHCC can increase the activation of antigen presentation by macrophages and the inhibition of tumor-derived immunosuppressive factors, enhance macrophage proliferation and activation, promote the differentiation of Th1 cells; increase the production of IL-12 by macrophages, increase the activity of NK cells; promote apoptosis of cancer cells. It has been indicated that AHCC increases TNF-α, interferon-γ and interleukin-12 and decreases the immunosuppressive acidic protein (IAP) and tumor growth factor (TGF) -α. Given its possible effects on the immune system, AHCC can be used to support cancer treatment to improve some of the negative side effects of chemotherapy.

As used herein, the term "intact protein" refers to a protein that preferably has not been subjected to chemical or enzymatic hydrolysis and is in a form substantially similar or identical to its natural state. In accordance with the present invention the "intact protein" can be chosen at least among casein, whey protein, soy protein, collagen or wheat protein.

In the context of the present invention the term "protein source" includes any amino acid-based proteinogenic matter, such as intact or hydrolyzed dietary protein, as well as added peptides or free amino acids and mixtures thereof, for example.

The protein source may include high-protein hydrolysates of hydrolysis prepared from animal and vegetable proteins treated with acid or enzymes, such as casein hydrolysates, whey, casein / whey, soy and mixtures thereof. By "high degree of hydrolysis" protein hydrolysates, it is understood that the intact protein is hydrolyzed into peptide fragments, most of which have a molecular weight of less than 1000 Daltons. Preferably, at least 75% (more preferably at least 95%) of the peptide fragments have a molecular weight of less than about 1000 Daltons. Free amino acids and synthetic short peptide chains can also replace protein hydrolysates or be added to them as a source of nitrogen, provided that the nutritional composition has an appropriate amino acid profile to the population to which it is targeted, as is known to the specialist in The field of nutritional formulations.

In a preferred embodiment of the immunonutritional compositions according to the present invention, the protein source may be of animal or plant origin. Therefore the protein source may include a combination of whey protein, casein or soy protein and its hydrolysates.

The whey protein source can come from natural whey, intact unhydrolyzed whey, whey concentrate, whey protein isolate or hydrolyzed whey protein.

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Casein can be supplied in free form or in the form of salt, for example sodium salt. It is also possible to supply casein in the form of calcium or potassium salt.

As used herein, the term "amino acids", if not indicated otherwise, refers to amino acids in free form and / or salt form chosen from at least one of the essential amino acids, eg isoleucine, leucine, lysine , methionine, phenylalanine, threonine, tryptophan, valine or histidine; conditionally essential amino acids, eg tyrosine, cysteine, argentine or glutamine, or non-essential amino acids, eg glycine, alanine, proline, serine, glutamic acid, aspartic acid, asparagine, taurine or carnitine. The role of amino acids in immune function is reviewed by Peng Li and colleagues in British J. Nutr., 98 (2): 237-252, 2007.

The present invention also relates to immunonutritional compositions that also include branched chain amino acids (AACR), eg valine, leucine, isoleucine or mixtures thereof, in free form and / or in saline form and / or in the form of protein. intact The AACR can be found in its three forms, as dipeptides, tripeptides, polypeptides, as a protein rich in AACR and / or as a manipulated protein to enrich the content of AACR. The dipeptides, tripeptides and polypeptides may include two or more AACRs. The nutritional products according to the present invention can similarly include precursors and / or metabolites of AACR.

Immune cells incorporate AACR into proteins and are capable of oxidizing AACR. The function of the immune system is to protect the host from agents, pathogenic infections and other pernicious aggressions. After infection there is a marked increase in the demand for substrates by the immune system; These substrates provide energy and are the precursors of the synthesis of new cells, effector molecules and protective molecules. Studies have indicated that AACRs are absolutely essential for lymphocytes to synthesize protein, RNA and DNA, and divide in response to stimulation. In experiments with mice, restriction of AACR in the diet impairs various aspects of immune function and increases susceptibility to pathogens. Post-surgical or septic patients who received intravenous forms of AACR showed better immunity, that is, better results. AACRs are therefore absolutely essential for lymphocyte responsiveness and are necessary to support other immune cellular functions.

AACRs can also promote glutamine synthesis and stimulate the Th1 immune response, a type of adaptive immune response. Long-term intense exercise has been linked to immunosuppression, which in turn affects natural killer cells, lymphokine activated lymphocyte cells and lymphocytes. It has been indicated that glutamine is an important food for macrophages and lymphocytes, and that it has immunity-stimulating effects. Its contribution as an oral supplement after exercise has clear beneficial effects in case of subsequent infections in athletes. However, the concentration of glutamine in plasma decreases after exertion, eg after a period of exercise. However, the reducing effect of glutamine concentration was neutralized by AACR supplementation, which was followed by a greater proliferative response in peripheral blood mononuclear cells. Supplementation with AACR stimulated the production of IL-2 and INF after exercise and a more pronounced decrease in the production of IL-4, indicating a deviation towards a Th1 immunological response. Supplementation with AACR was also effective in keeping the concentration of glutamine in plasma constant. Bassit, R. A. et al., Nutrition, 18 (5): 376-379, 2002.

In addition to improving metabolic parameters, oral supplementation enriched with AACR can improve morbidity and quality of life of patients undergoing major liver resection and chemoembolization. However, the role of AACRs as nutritional support for stressed surgical patients and cancer patients is not yet clearly defined, despite their potentially beneficial biological properties. Choudry, H.A. and others, J. Nutr., 136 (1 Suppl.): 314S-8S, 2006.

The immune response requires higher amounts of AACR. In fact, stimulated lymphocytes show greater absorption of AACR for cell expansion, including leucine, isoleucine and valine. In addition, leucine is an activator of the mTOR signaling pathway, which regulates protein synthesis and degradation and also antagonizes the autophagic process of cells suffering from stress or starvation. The AACRs are incorporated into the nutritional compositions of the present invention in amounts comprised approximately between 2 and 30 g daily, preferably in an approximate amount of 3 g daily.

The nutritional compositions of the present invention may further comprise glutamine (Gln) and / or arginine and / or citrulline and / or branched chain amino acids (AACR).

Glutamine is a primary nutrient substrate for immune system cells. In addition to being a fundamental source of glutamate, Gln regulates glutathione synthesis and is a precursor to the purine and pyrimidine nucleotides necessary for lymphocyte proliferation. In its role as an anticancer agent, Gln is capable of increasing the innate cytolytic activity of NKs, killer dendritic cells and macrophages. Gln also contributes to the antigen-specific cytolytic action of CD8 + T cells against tumor cells.

Glutamine may be in the form of an added amino acid. In the context of the present invention "added amino acid" refers to a non-protein-linked amino acid that is incorporated apart from typical dietary sources of protein such as milk, meat and vegetable proteins. The added amino acid may be present in the form of a

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free amino acid and / or of a di-and / or tripeptide comprising the amino acid. For example, glutamine can be added in the form of a dipeptide such as L-alanyl glutamine. Free glutamine is not stable in a liquid environment; therefore, if the composition must be sold in liquid form, glutamine will have to be added as a dipeptide or other stable form in liquid medium. In case of having to supply the composition in liquid form, another possibility would be to include an appropriate amount of glutamine powder as a module for mixing with the liquid immediately before consumption.

The amount of glutamine may vary between approximately 5 g and 30 g daily, more preferably between approximately 6 g and 9 g daily.

In addition to the above, Gln can increase HSP expression in normal bowel epithelial cells. Expression of HSP in tumor cells during anticancer treatment may result in an increase in immunogenicity of tumor cells. The anticancer treatment induces stress in the tumor cells, which in turn increases the effectiveness of the innate immune system to contribute to the cytotoxic effect on the transformed cells and acts in conjunction with the drugs in the removal of tumor mass. The amount of Gln is preferably about 5 g to 30 g, more preferably about 6 g to 9 g.

Arginine is synthesized from citrulline as an immediate precursor in many tissues, but more importantly in the kidney. In turn, citrulline is synthesized from glutamine, glutamate and proline at the intestinal level. The levels of citrulline and arginine in the plasma decrease markedly during malnutrition, fasting, in different types of lesions, when there are tumors, during anticancer treatment and sepsis. It has been thought that this contributes to the existing immunodeficiency in cancer.

The biological activities of arginine on immune function could be categorized as direct and indirect. Therefore it can be assumed that citrulline will also produce the same effects as arginine as a result of its role in arginine synthesis.

Many direct activities on the immune system are related to the function of T cells and are mainly explained by the induction of the expression of one of the components of the T-cell receptor. In fact, the physiological levels of arginine (150 µM) modulate the ξ chain of the T cell receptor necessary for the function of T cells. Importantly, citrulline has been shown to act synergistically with arginine to express the ξ chain of CD3, prolonging the half-life of its mRNA.

There are several types of tumors that express arginase or induce arginase expression in tumor cells, resulting in one of the fundamental mechanisms of immunodeficiency normally observed in host-tumor interaction. Immunodeficiency affects the antigen-specific cytotoxic function of CD8 and also the innate cytotoxicity of NKs and macrophages for transformed cells. The macrophages associated with the tumor have a direct participation in the immunosuppressive process by producing arginase and also expressing a phenotype capable of inducing regulatory T cells that prevent the cytotoxic action of the immune system. All these observations support the claim that simultaneous administration of citrulline and arginine can compensate for immunodeficiency during antitumor activity.

The metabolism of L-arginine in myeloid suppressor cells is critical to inhibit the activation of T cells (Bronte, V. et al., Nat. Rev. Immunol., 5: 641-654, 2005). Different metabolic pathways have been described in MSCs that explain the increased consumption of arginine and the deprivation of this amino acid for T cells, a prerequisite for the activation of T cells. Alternatively, activated macrophages are characterized by increased expression. of arginase, an enzyme responsible for the decrease of arginine.

The daily dose of arginine included in the immunonutritional compositions of the present invention may vary approximately between 5 g and 30 g daily, preferably in an approximate concentration range of 10 g to 15 g daily.

The daily dose of citrulline included in the immunonutritional compositions of the present invention may vary approximately between 1 g and 30 g daily, preferably in an approximate concentration range of 2 g to 15 g daily.

In several clinical applications of citrulline supplementation the efficacy of a dose of three to four grams taken twice daily has been demonstrated. After administration the results usually occur over a period of 3-5 days. Returning now to part of the prior technical state, U.S. Pat. No. 5,576,351 generally describes the treatment of a deficient immune response by administering arginine or ornithine or mixtures thereof to humans suffering from or at risk of suffering a defective immune response. However, there is no disclosure that any benefit is obtained in mitigating or alleviating the effects of such conditions by administering arginine.

WO / 2007/114903 offers a method and formulation for the treatment or maintenance of conditions that would benefit from the increase or preservation of arginine levels in the blood and that have

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With regard to vitamin E, it has been indicated that the treatment of cancer patients with high daily doses of vitamin E in the short term may enhance the action of NK cells. The amount of vitamin E administered to cancer patients was about 750 mg daily for two weeks. Hanson, M.G. and others, Cancer Immunol. Immunother., 56 (7): 973-984, 2007. Short-term treatment with vitamin E significantly improved the cytolytic action of NK cells. The increased activity of NK cells among patients' peripheral blood mononuclear cells was not due to an increase in the number of NK cells or the percentage of subpopulation of NK CD56 cells (dim). Likewise, treatment with vitamin E was related to a small but constant induction of NKG2D expression among all patients studied. Tumor-induced immune suppression is not limited to the adaptive T-cell system and the functional defects of dendritic cells (DC) and NK. Vitamin E has the ability to increase the production of cytokines IL-2 and IFN-gamma by Th1 and increase the activity of NKs through a mechanism probably very different from that of histamine. Hanson

M.G. and others, cited above.

Proteins are milk proteins (whey or whey protein in combination with casein) and amino acids that provide approximately 20-40% of the energy content of the product, preferably about 30% of the energy content of the product. Proteins may also include soy, casein and hydrolysates.

The lipid source may comprise saturated fatty acids (AGS), monounsaturated fatty acids (AGMI) and / or polyunsaturated fatty acids (AGPI). AGS can be found in part as medium chain triglycerides (TCM). As discussed here, TCMs refer to triglycerides comprising C6-C12 fatty acids. All fatty acids from the lipid source can be found in the form of n-3 fatty acids. The n-3 fatty acid is preferably chosen from α-linolenic acid (18: 3n-3), eicosapentanoic acid (AEP, 20: 5n-3), docosapentanoic acid (ADP, 22: 5n-3) and docosahexaenoic acid (ADH , 22: 6n-3) and mixtures thereof.

Lipids can provide 25 -40% of the energy content of the product, preferably 30% of the total energy of the product, 50% of which is constituted by medium chain triglycerides. Polyunsaturated fatty acids (eg eicosapentanoic acid (AEP) and docosapentanoic acid (ADP)) are from vegetable oils or from fish oil, with a n6: n3 ratio range of less than 6, preferably equal to 2-3 approximately.

It has been seen that essential fatty acids (AGE) are involved in the modulation of lymphocyte reactivity and in the destruction of several tumor cells in vitro. Purasiri, P. and others, Eur. J. Surg. Oncol., 21 (3): 254-260. In short-term oral supplementation (15 days) with essential fatty acids (AGEs), AGEs did not significantly alter the cytotoxic activity of NK and LAK cells in patients with localized cancer. However, in the group with advanced disease, the reduction in cytotoxic activity of NK and LAK cells occurred on day 15 and decreased continuously, reaching minimum levels 6 months after supplementation. In the group with advanced cancer there was no change in the cytotoxic activity of the NK and LAK. However, long-term supplementation can have detrimental effects on natural anti-cancer cytotoxic mechanisms in patients with malignant disease. Purasiri, P. and others, cited above.

Examples of ácidos-3 fatty acids include AEP and ADH. Both AEP and ADH give rise respectively to eicosanoids and docosanoids, which may have different properties from eicosanoids derived from arachidonic acid. The AEP and ADH lead to the formation of resolvins. Calder, P.C. and others, Prostaglandins Leukot. Essent Fatty Acids, 77 (5-6): 327-335, 2007. On the other hand it is known that resolvins reduce cellular inflammation by inhibiting the production and transport of inflammatory cells and chemicals to the sites of inflammation. Resolvins have an immune role in the kidneys as a remedy against acute renal failure. Serhan, C. N. et al., J. Exp. Med., 196 (8): 1025-37, 2002.

The greater incorporation of AEP into the phospholipids of immune cells can increase the production of eicosanoids derived from AEP such as prostaglandin E3 (PGE3) and leukotrienes (LTs) of the 5 series, since the AEP can serve as a substrate for cyclooxygenase and lipoxygenase enzymes. A greater generation of LTs of the 5 series has been proven using macrophages from mice fed with fish oil, neutrophils from human subjects infused for several days with lipid emulsions containing fish oil, and neutrophils from humans supplemented for several weeks with fish oil by mouth.

Based on the above, fatty acids fulfill a series of functions in immune cells. They can constitute a reserve for the generation of energy, they can act as components of the phospholipids of the cell membrane contributing to the physical and functional properties of these membranes, as covalent modifiers of the protein structure with influence on the location and cellular function of the proteins, as regulators of genetic expression by effect on the activity of the receptors, in the processes of intracellular signaling or in the activation of transcription factors, and as precursors for the synthesis of bioactive lipid mediators such as prostaglandins (PGs), leukotrienes (LTs), lipoxins and resolvins.

Changes in the fatty acid composition of membrane phospholipids can influence the activity of immune cells, as illustrated below, including the following steps: (1) property alterations

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membrane physics such as their order and structural domain; (2) altered effects on cell signaling pathways, either by a change in the expression, activity or avidity of membrane receptors or by modification of the transduction mechanisms of intracellular signals; and (3) alterations in the pattern of lipid mediators (PGE2). As a result of these different changes, the activation of the transcription factor is altered and the genetic expression is modified. Different mediators can produce different activities and biological powers. Calder, P.C. and others, cited above.

Figure 1

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Carbohydrates can provide an energy content in the range of about 30 to 50% of the product, preferably about 40%.

The source of carbohydrates can consist of any suitable and digestible carbohydrate or mixture of carbohydrates. For example, the source of carbohydrates can be maltodextrin, natural or modified tapioca starch, corn, rice, other cereals, potatoes, or starch high in amylose, sucrose, glucose, fructose and / or mixtures from them.

The immunonutritional compositions according to the present invention may be clinically free of lactose. In the context of the present invention, the term "clinically lactose-free" refers to immunonutritional compositions having a maximum of 0.2 g of lactose per 100 kcal of the composition. The composition preferably has less than 0.2, more preferably less than 0.17 g of lactose per 100 kcal of the composition.

The immunonutritional compositions according to the present invention may also be gluten free.

The immunonutritional compositions according to the present invention may also carry other nutritional supplements, for example vitamins, minerals, trace elements, as well as additional sources of nitrogen, carbohydrates and fatty acids. They can be added to the patient's oral intake or supplied in the form of a single complete nutritional formulation that provides all the basic daily amounts of vitamins, minerals, carbohydrates, fatty acids and other necessary ingredients.

The immunonutritional compositions according to the present invention can be formulated suitably for enteral administration. They are especially suitable for enteral use, such as oral and / or tube administration. These compositions are conveniently administered in the form of an aqueous liquid. The compositions according to

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The present invention which are suitable for enteral application are therefore in aqueous form or a powder that must be mixed with water before use. In the case of tube feeding, the amount of water added will depend on the need for liquid and the patient's condition.

The term "pharmaceutically acceptable salt" refers to salts that, within the scope of reasonable medical judgment, are suitable for use in contact with human tissue without causing an inappropriate response of a toxic, irritating, allergic and similar type, and have a reasonable benefit / risk ratio.

As applied here, the numerical intervals are intended to include any number and subgroup of numbers contained in the interval, whether they are specifically disclosed or not. In addition, these numerical intervals should be interpreted as the basis for a claim addressed to any number and subgroup of numbers in this range. For example, a disclosure of 1 to 10 should be interpreted as an interval that includes limits of 2 to 8, 3 to 5, 6, 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, and so on.

All references to particular features or limitations of the present invention include the respective plural feature or limitation and vice versa, unless otherwise specified or the context in which the reference is made clearly implies otherwise.

As used herein, all combinations of stages of the method or process may be performed in any order, unless otherwise specified or the context in which reference is made to the combination clearly implies otherwise.

As used herein, all percentages, parts and proportions refer to the weight with respect to the total composition, unless otherwise specified. As all weights of the listed ingredients are based on their active level, they do not include solvents or by-products that may be contained in commercially available materials, unless otherwise specified.

The compositions and methods of the present invention may comprise, consist of or consist essentially of the basic elements and limitations of the present invention described herein, as well as any additional or optional ingredient, component or limitation described herein or that may be useful in compositions and methods of the general type described herein.

"Treatment" refers to the administration of medicines or compositions or formulations or to the performance of medical procedures with respect to a mammal, including a human, for the prophylaxis (prevention), cure, improvement or normalization of the disease, disease or insufficiency of the afflicted or deficient patient.

"Patient" or "subject" refers to a human or non-human mammal that can benefit from the nutritional composition and the method described in the present patent application.

A "therapeutically effective amount" or a "nutritionally effective amount" is an amount of an agent, composition or formulation that is sufficient to achieve the desired treatment effect.

"Parenteral" refers to the route of materials through or basically through the epidermal layers of the human body, usually by intravenous (IV), intramuscular (IM) or subcutaneous (SC) means.

As used herein, the term "enteral" refers to administration through the alimentary tract. The specialist knows that this administration can take place in the intestine, which is the duct that goes from the stomach to the anus and is divided into small intestine and large intestine, through the mouth, through a nasogastric tube into the stomach and by other means known in the technical state.

“Pharmaceutically acceptable” means approved by a federal government or state government regulatory agency, or listed in the U.S. Pharmacopoeia. or in another generally recognized pharmacopoeia, for use in animals and more specifically in humans.

"Vehicle" refers to a diluent, adjuvant, excipient or support with which the therapeutic agent is administered. These pharmaceutical vehicles can be sterile liquids such as water and oils, including those of petroleum and those of animal, vegetable or synthetic origin, for example peanut, soy, mineral oil, sesame oil and the like. Saline solutions and aqueous solutions of dextrose and glycerin can also be used as liquid vehicles, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, crete, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, glycol , water, ethanol and the like.

As used herein, the term "cancer therapy" refers to chemotherapy, surgery, radiation, gene therapy, immunotherapy, biological therapy, differentiating agents, chemopreventive agents or a combination thereof. In some embodiments, chemotherapy refers to drugs or cytotoxic agents.

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As used herein, the term "chemotherapy" refers to a process of eliminating proliferating cells by a cytotoxic agent. The term "during chemotherapy" refers to the period of duration of the effect of the cytotoxic agent administered. On the other hand, the expression "after chemotherapy" means that all situations in which a composition is administered after a cytotoxic agent are covered, independently of any previous administration thereof and also independently of the persistence of the effect of the administered cytotoxic agent.

If the method of the present invention is applied to chemotherapy, at least one immunonutritional composition may be administered before, during or after chemotherapy (ie before, during or after administering a cytotoxic agent). For example, the immunonutritional compositions of the present invention may be given to the subject ten to three days before a chemotherapy cycle (pre-chemotherapy or before chemotherapy) up to ten to seven days after the cycle (post-chemotherapy or after chemotherapy).

Examples of sweeteners include, but are not limited to, sodium saccharin, aspartame, stevioside, stevia extract, para-methoxycinnamic aldehyde, dihydrocalcone neoesperidine, routine knob and the like.

As usual dosage forms of pharmaceutical products it is possible to mention, without limitation, oral preparations (liquid preparations such as extracts, elixirs, syrups, tinctures and lemonades; solid preparations such as capsules, granules, pills, powders and tablets), injections , infusions, nasal drops, eye drops, suppositories, aerosols and dosage forms for percutaneous administration such as ointments and patches.

In accordance with the present invention, the compositions according to it can be offered in the form of dietary products, eg as supplements or in the form of a nutritional formulation, eg a medicinal product for eating

or drink, eg in the form of a complete or partial meal, as a food additive or dissolving powder, or in the form of a pharmaceutical formulation, eg in the form of a tablet, pill, envelope or capsule.

In another aspect the present invention provides medicinal product for eating or drinking, a dietary or nutritional supplement or a pharmaceutical formulation comprising the immunonutritional compositions of the present invention.

The compositions of the present invention in the form of dietary products, eg supplements or pharmaceutical formulations, may consist exclusively of the compositions of the present invention and optionally pharmaceutically or nutritionally acceptable carriers.

The compositions of the present invention may be in the form of medicinal products for eating or drinking, eg in the form of powders for dissolution. The powder can be combined with a liquid, eg water or other liquid such as milk or fruit juice, eg in a proportion of about 1 to 5 powder to liquid, to obtain a ready-to-drink composition or a beverage snapshot.

Optionally the compositions of the present invention can be nutritionally complete, that is, they can include vitamins, minerals, trace elements, as well as sources of nitrogen, carbohydrates and fats and / or fatty acids, so that they can be used as a single source of nutrition that basically provides all the indispensable daily amounts of vitamins, minerals, carbohydrates, fats and / or fatty acids, proteins and the like. Accordingly, the compositions according to the present invention can be supplied in the form of a nutritionally balanced complete meal, suitable for example for oral or tube feeding, eg through nasogastric, nasoduodenal, tracheoesophageal, gastrostomic or jejunostomic tubes, or for peripheral nutrition

or total parenteral. The compositions of the present invention are preferably for oral administration.

The present invention provides means to assist the immune system during anticancer treatment by chemo or radiotherapy.

The present invention provides methods to take advantage of increased tumor cell expression of stressed cells ("danger signal") by inducing the recognition and elimination of these cells by innate immune cells such as natural killer cells (NK), natural killer cells T (NKT), macrophages (Macs) and killer dendritic cells (KDC). Innate immune cells are strongly activated by finding "warning signs" in tumor cells during anticancer treatment.

The following examples describe the presence of suppressive immune cells and the immunological activity of animals that have tumors and suffer a deterioration of their innate and adaptive immune response, whether or not they undergo chemotherapy. An example is also provided describing the beneficial effects of immunonutrition in mice that have tumors and undergo antitumor therapy. Five examples of immunonutritional compositions are also given below, which vary from one to another by the type and amount of immunity enhancing agents therein.

Example 1

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Transient lymphopenia included CD3 and CD 19 (B cells), NKs; (lymphocyte subgroups); Peripheral blood carries a minority of dendritic cells and monocytes.

Tumor growth could be observed 5-6 days after implantation s.c. of the cells. The size of the

5 tumor does not show a significant change after chemotherapy, but again growth is observed from 8-10 days after chemotherapy. Then the tumor increase continues until sacrifice. In control mice, which had a tumor but were not treated with chemotherapy, the growth rate is higher until the end of the trials (animal sacrifice).

10 Figure 2 illustrates how the adaptive immune response is stimulated by the immunogenicity promoted by chemotherapeutic treatment. Chemotherapy damages cancer cells and therefore increases their susceptibility to the immune system. In Figure 2, the divergence at day 14 of the two treatment groups (oxa-10; oxa-12.5) with respect to the control group is related to the enhanced immune response. Although the tumor continues to grow, it does so at a slower rate than the control group (without chemotherapy).

15 Figure 2. Antitumor effect of oxaliplatin in a murine model implanted with tumor versus control

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20 Example 2

Presence of immunosuppressive cellular mechanisms in animals that have tumors and are undergoing chemotherapy - state of the innate and adaptive immune response

25 mice In the trials, eight weeks old C57BL / 6 (H-2b) inbred mice were used. The animals were distributed in 7 different dietary groups. There was a control group that received the AIN 93 diet for adult rodents (maintenance). The test diets were administered in appropriate doses to the animal model: (a) the control diet was supplemented with 1% (w / w) of L-arginine, (b) 25% of the protein was replaced by glutamine, ( c) 1% (w / w) L-citrulline, (d) 1 g / kg body weight with correlated active hexose compound, (e) 20 mg / day of

30 nucleotides of RNA and (f) 25 mg / day of lactoferrin. One week later the mice were inoculated subcutaneously (s.c.) on the left flank with 1x106 tumor cells and tumor growth was monitored every 2 days by measuring it with a caliber. After 6 days after tumor inoculation, the animals were treated with oxaliplatin or doxorubicin. Tumor growth was monitored every two days after chemotherapeutic treatment and the mice were sacrificed two weeks after such treatment. Control animals without chemotherapeutic treatment

35 were tested in parallel with all diets tested. Body weight was determined every two days. Blood samples were taken on days 2, 4 and 10 after chemotherapeutic treatment and the day of sacrifice (14 or 28 days). An autopsy was performed and the tumor mass was evaluated.

Cancer cell lines The cell line of sarcoma induced by methylcolantreno (MCA) that expresses the

Ooalbumin exogenous antigen (OVA) was cultured in DMEM or RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 µM 2-mercaptoethanol, 150 U / ml streptomycin, 200 U / ml penicillin and SBF 10% heat inactivated. 1x106 tumor cells were injected into the flank of the mice 6 days before the chemotherapeutic treatment.

45 Hematological evaluation. On days 2, 4, 10, 14 and 28 the red blood cell counts, hemoglobin were carried out

and hematocrit.

White blood cell counts and leukocyte differential formula were examined at the same times. Blood samples were also used to determine immune cell populations.

5 Cytometric flow analysis. The analysis of the cellular subgroups CD11c +, CD11b + Gr-1 + and CD11b + Gr-1-, CD14 +, CD19 +, CD16 +, CD56 +, CD3 +, CD8 +, CD4 + was performed. The antibody battery used allowed the study of the subgroups of B and T cells, NK, NKT cells, macrophages, dendritic cells, granulocytes.

10 Evaluation of tumor growth. Tumor growth was monitored every 2 days using calipers and tumor volume was calculated by applying the formula length x width x width x 0.52 mm3.

Results

15 All the diets tested gave rise to a similar weight gain curve during the 8 days prior to tumor transfer. After inoculation s.c. of tumor cells in mice, tumors need 5 to 6 days to start growing, as seen with the caliber. After implantation of the tumor cells and before chemotherapy, no change in tumor weight was observed. The animals lost weight during the first days after chemotherapy. Maximum weight loss was reached between days 4 and 10 after

20 of the chemotherapy and then the animals remained stable weight or even began to recover body weight. No differences were observed between the different diets.

The maximum weight loss was around 10 percent for all doses tested for doxorubicin (2.5, 5, 7.5 and 10 mg / kg) and about 15 percent for the maximum dose of oxaliplatin (10 mg / kg Other doses tested 25 were 5 and 7.5 mg / kg).

Toxicity to red blood cells evaluated by the number of erythrocytes, hemoglobin and hematocrit levels showed a different pattern. Both chemotherapeutic agents induced a decrease in red blood cells that reached the lowest levels between days 6 and 10 after chemotherapy and did not reach stable levels until day 16. The

30 diet supplemented with arginine avoided the marked drop in erythrocytes observed between days 6 and 10 (Figure 3). This group was different from the control group and also from the other treatments. In addition, the combination of arginine with chemotherapeutic treatment further reduced the size of the tumor compared to chemotherapy applied without more (Figure 4).

35 Figure 3. Effect of arginine dietary supplementation on reducing bone marrow toxicity, compared to the control diet

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40 Figure 4. Arginine in the diet together with cancer therapy reduces tumor size compared to the control diet

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White blood cell counts decreased in the first week after chemotherapy. Before the

5 tenth day white blood cell counts begin to recover and exceed the original baseline after day ten and tend to remain high until the end of the experiment. Control animals not treated with chemotherapeutic agents have a more stable level of white blood cells during the experiment, which tends to rise after day 15 (Figure 5). In animals treated with oxaliplatin, the leukocyte increase tended to be higher than in the group that received the diet supplemented with lactoferrin.

10 Figure 5. Effect of oxaliplatin on the white blood cell population

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15 Flow cytometry studies of leukocytes and subgroups of immune cells showed that the overall decrease in lymphocytes was caused by chemotherapy about 10 days after treatment. The loss of CD3 + cells was partially modulated in animals that received the diet supplemented with arginine. The global lymphocyte population decreased less after chemotherapy in the groups that received the amino acids glutamine and citrulline, as well as lactoferrin. In the treatment group that received dietary nucleotides, it was observed

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until day 55 when animals are slaughtered. Peripheral blood cells were studied 10 days after the first and second cycle and before sacrifice. Neutrophil counts give 43% of the average values recorded the day before starting chemotherapy in the control group and 70% in animals receiving the test diet supplemented with immunonutrients. There are no differences in tumor regression between the two dietary groups. In both, a reduction in tumor mass is measured. The histological study of the remaining tumor lesions and of the dysplastic lesions shows a similar mitotic activity of the cells that pass through the cell cycle (labeling index with PCNA),

Treatment of toxic effects caused by both cancer therapy and bone marrow tumor and immune compartments

The maintenance of immunocompetence during cancer treatment increases the body's ability to identify and destroy cancer cells in the body. As a result, any attack on the compartments involved in maturation or in the maintenance of the immune system increases the danger of cancer progression. Treatment with chemicals or radiology is designed to destroy cancer cells and some of them are very effective in reducing the growth rate of tumors (Figure 8).

Figure 8. Antitumor effect of oxaliplatin in a murine model implanted with tumor

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The deceleration of the growth of the tumor, or even the reduction of its size, through the aggressive use of chemo- and / or radiotherapy is part of the neoadjuvant strategy prior to surgical intervention. However, anti-cancer therapies also affect other rapidly dividing cells that are produced, for example, by the bone marrow and used to kill cancer cells.

Since the bone marrow is the place where blood cells form, toxicity (for whatever reason) causes a deficiency of blood cells. A result of this toxic effect on the bone marrow may be a life-threatening infection, because the body cannot produce leukocytes in response to invading bacteria and viruses. In addition, toxicity causes anemia due to the low number of red blood cells and even severe bleeding caused by a lack of platelets.

As described above, cancer cells damaged by neoadjuvant treatment can express components recognized by the immune system, but the body can only organize a defense when the immune system is not so severely diminished by anticancer therapy itself. Therefore, immunocompetence must be maintained, reducing bone marrow toxicity, to increase the effectiveness of the treatment. The "opportunity interval" for the immune system to regain control of the transformed cells and suppress the remaining tumor cells occurs in the days following chemotherapy. To take advantage of this period of increased antigenic or immunogenic expression, the present invention describes methods (nutritional and other) that can enhance the innate immune response and the antitumor immune response. The selective use of nutrition (but also of pharmaceutical compounds) to condition the immune system before, during and after the cycles of chemo-and radiotherapeutic treatment can correct the effects of acute immune toxicity induced by these cancer therapies.

Our data show that cancer therapy creates an initial attack to the bone marrow and therefore also to

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Claims (1)

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